1. Field of the Invention
This invention relates to a method for producing peptides, which have an activity of an agonist of luteinizing hormone releasing hormones (LHRH) secreted from the hypothalamus or their salts. The present invention further relates to an intermediate peptide, its production method, its crystals and a method for producing the crystals.
2. Description of the Related Art
U.S. Pat. No. 4,008,209 which corresponds to Japanese Patent Application Laid-open No. 50-059370/1975, describes the following method for producing a peptide of the formula: (Pyr)Glu--His--Trp--Ser--Tyr(or Phe)--X--Leu(or IIe, or Nle)--Arg--Pro--NH--R (SEQ ID NO: 13), wherein each amino acid residue has L-configuration unless otherwise indicated; X represents D--Leu, D--NLe, D--NVa, D--Ser, D--Abu, D--Phg, D--Phe or .alpha.--Aibu; R represents an alkyl group which may optionally have hydroxyl. ##STR1##
In the reaction scheme, all symbols have the same meanings as defined above.
Japanese Patent Application Laid-open No. 51-6926/1996, which corresponds to U.S. Pat. No. 3,997,516, describes a method for producing a peptide having a guanidino group which comprises protecting the guanidino group of a guanidino group-containing peptide with a lower alkoxybenzenesulfonyl group or tri(lower)alkyl-benzenesulfonyl group.
Furthermore, Japanese Patent Application Laid-open No. 51-100030/1996, which corresponds to U.S. Pat. No. 3,997,516, describes a method for producing a peptide having a guanidino group which comprises protecting the guanidino group of a guanidino group-containing peptide with a lower alkoxybenzenesulfonyl group or tri(lower)alkylbenzenesulfonyl group and, after the peptide condensation reaction, removing the protective group with a halogenosulfonic or lower alkylsulfonic acid or a Lewis acid.
For the production of a peptide on a commercial scale, it is essential that various parameters such as (1) qualities of starting materials, (2) production cost, (3) workability, (4) safety to operators, and (5) prevention of pollution should satisfy certain practically acceptable levels and every process that is satisfactory at the laboratory level is not necessarily satisfactory at the factory level. Therefore, design of a commercial process for producing a peptide involves many problems that must be solved for satisfying a variety of requirements such as the modality of peptide chain extension; the selection of the site of fragment condensation; the method for inhibiting isomerization at condensation of fragments; the selection of protective groups for the .alpha.-position and side-chain functional groups; the method for final elimination of such protective groups; the method for purification of the end product peptide; and the overall operability of the series of steps, among others.
Furthermore, a diversity of methods and a variety of reaction conditions may be contemplated for the synthesis of peptides in general but it is often the case that because of non-crystallizability, many intermediates used in the process cannot be sufficiently purified or require time-consuming fractionation procedures, with the result that many processes are not satisfactory in the reproducibility of quality and yield. Thus, the physical characteristics such as crystallizability, stability, and solubility of intermediates, which are key factors in the respective production stages, hold sway over whether a process can be commercially acceptable in many cases.
Referring to the methodology for production of the peptide of the formula:(Pyr)Glu--His--Trp--Ser--Tyr(or Phe)--X--Leu(or Ile, or Nle)--Arg--Pro--NH--R (SEQ ID NO: 13), the process disclosed in U.S. Pat. No. 4,008,209, which corresponds to Japanese Patent Application Laid-open No. 50-059370/1975, protects the guanidino group of Arg with nitro and, therefore, the group Z used for protecting the .alpha.-amino group can hardly be reductively eliminated selectively, so that HBr--AcOH is used for removal of group Z. Inevitably, in this process, benzyl bromide, which is highly lacrimetory, is by-produced in a large quantity and, moreover, a large amount of ether must be used for isolation of the end product. In addition, while the end product of this reaction is usually recovered in the hydrobromide form, the hydrogen bromide must be removed with, for example, an ion exchange resin in order that the objectionable isomerization on condensation of fragments may be inhibited. Furthermore, while the gradient elution method is used in the chromatographic purification of the final end product, the above peptide, the lower reproducibility of the concentration gradient necessitates rigorous qualitative testing, with the result that the operation for steady production of the desired product of uniform quality cannot be stand-ardized. Therefore, the process is not suited for industrial-scale production.
Furthermore, in the U.S. Pat. No. 3,997,516, a reaction scheme produces a peptide of the formula: (Pyr)Glu--His--Trp--Ser--Tyr--D--Leu--Leu--Arg(MBS)--Pro--NH--C.sub.2 H.sub.5 (SEQ ID NO: 14) wherein MBS denotes p-methoxybenzenesulfonyl group. However, in the U.S. patent, only the reaction scheme is described, but other details such as reaction conditions are not described at all.
Thus, an industrially profitable process for producing peptides which have an activity of an aqonist of LHRH or its salts with safety, expedience, high yield and good reproducibility has not yet been developed.
The inventors of the present invention explored the above-mentioned problems with diligence and succeeded in establishing a protocol for the effective production of intermediate peptide (II), mentioned below, through inhibition of isomerization of amino acid residues in the hydrolysis reaction step, a protocol for crystallization of the peptide (II); and deprotection on an industrial scale of peptide (I'), mentioned below; and a protocol for industrial-scale purification of the objective peptide (I), mentioned below. Accordingly, the inventors arrived at a process for producing said peptide (I) with safety, high yield, and good reproducibility, and have completed the present invention.